I. Genetic Engineering Involves Four Basic Steps

A. Because the genetic code is universal, bateria can transcribe and translate human genes, e.g. the human gene for the protein hormone, insulin

B. 1973, Cohen & Boyer: inserted frog rRNA gene into E. coli; later won Nobel Prize

C. Genetic engineering: manipulating genes for practical purposes

D. Recombinant DNA: DNA which is a combination of the DNA from two different organisms

E. One example of genetic engineering:

Step 1. Cutting DNA

• using restriction enzymes: bacterial enzymes which cut DNA at specific nucleotide sequences
• vector: an agent used to carry a gene from one organism into another
• plasmids: circular DNA from bacteria independent of the main bacterial chromosome
• thus, restriction enzymes are used to cut a gene of interest from one organism and splice it into a vector, such as a plasmid, which has been cut with the same restriction enzyme

Step 2. Making recombinant DNA

• the gene of interest is spliced into the plasmid, which now contains both bacterial genes and the gene of interest
• DNA ligase seals the gene of interest into the plasmid

Step 3. Transformation & Cloning

• add the recombinant plasmid to a bacterial colony
• the bacteria take up the recombinant plasmid by transformation
• allow the bacteria to reproduce by binary fission, making many copies of the gene of interest

Step 4. Screening

• eliminate the bacteria that did not take up the plasmid by transformation
• eliminate the bacteria whose plasmids do not contain the gene of interest

F. Cutting DNA and making recombinant DNA

• some restriction enzymes recognize palindromic sequences of DNA
• DNA cut by most restriction enzymes leaves sticky ends
• complementary sticky ends from the gene of interest and the plasmid allow them to hydrogen bond together
• DNA ligase creates covalent bonds, sealing the gene of interest into the plasmid

G. Cloning and screening bacterial cells

• finding and isolating the bacterial cells that actually contain the gene of interest is difficult
• by using plasmids that contain antibiotic resistance genes, screening by the addition of an antibiotic kills the bacteria that were not transformed

II. Southern Blots Confirm a Cloned Gene is Present

A. Southern blotting involves four steps to isolate which bacterial colonies have the gene of interest:

• Step 1. DNA from each bacterial colony is cut into a set of DNA fragments, of varying lengths, by restriction enzymes

• Step 2. DNA fragments are separated by gel electrophoresis, in which electricity draws the smaller fragments farther through a gel and larger fragments a shorter distance, thus producing a pattern of bands organized by size; a chemical is then added to separate the DNA fragments into single strands

• Step 3. DNA is transferred to filter paper (blotted), and a radioactive- or flourescent-labeled RNA probe, complementary to the DNA gene of interest, is added

• Step 4. Only the DNA fragments that contain the gene of interest will bind to the probe; thus, the bacterial colonies which contain the gene have been identified

B. the identified bacterial colonies that contain the gene of interest can then be used

C. the bacteria can be used to produce large quantities of the protein coded for by the gene of interest, e.g. insulin

D. the gene of interest can be sequenced and compared to similar genes in other organisms, determining their evolutionary relationships